Methods and compositions for treating brain diseases

ABSTRACT

The present disclosure provides targeting peptides and vectors containing a sequence that encodes targeting peptides that deliver agents to the brain.

RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/959,638, filed Jul. 14, 2007, the entirety of which isincorporated herein by reference.

FEDERAL GRANT SUPPORT

Portions of the present invention were made with support of the UnitedStates Government via a grant from the National Institutes of Healthunder grant number HD33531. The government has certain rights in theinvention.

BACKGROUND

Gene transfer is now widely recognized as a powerful tool for analysisof biological events and disease processes at both the cellular andmolecular level. More recently, the application of gene therapy for thetreatment of human diseases, either inherited (e.g., ADA deficiency) oracquired (e.g., cancer or infectious disease), has received considerableattention. With the advent of improved gene transfer techniques and theidentification of an ever expanding library of defective gene-relateddiseases, gene therapy has rapidly evolved from a treatment theory to apractical reality.

Traditionally, gene therapy has been defined as a procedure in which anexogenous gene is introduced into the cells of a patient in order tocorrect an inborn genetic error. Although more than 4500 human diseasesare currently classified as genetic, specific mutations in the humangenome have been identified for relatively few of these diseases. Untilrecently, these rare genetic diseases represented the exclusive targetsof gene therapy efforts. Accordingly, most of the NIH approved genetherapy protocols to date have been directed toward the introduction ofa functional copy of a defective gene into the somatic cells of anindividual having a known inborn genetic error. Only recently, haveresearchers and clinicians begun to appreciate that most human cancers,certain forms of cardiovascular disease, and many degenerative diseasesalso have important genetic components, and for the purposes ofdesigning novel gene therapies, should be considered “geneticdisorders.” Therefore, gene therapy has more recently been broadlydefined as the correction of a disease phenotype through theintroduction of new genetic information into the affected organism.

In in vivo gene therapy, a transferred gene is introduced into cells ofthe recipient organism in situ that is, within the recipient. In vivogene therapy has been examined in several animal models. Several recentpublications have reported the feasibility of direct gene transfer insitu into organs and tissues such as muscle, hematopoietic stem cells,the arterial wall, the nervous system, and lung. Direct injection of DNAinto skeletal muscle, heart muscle and injection of DNA-lipid complexesinto the vasculature also has been reported to yield a detectableexpression level of the inserted gene product(s) in vivo.

Treatment of diseases of the central nervous system, e.g., inheritedgenetic diseases of the brain, remains an intractable problem. Examplesof such are the lysosomal storage diseases. Collectively, the incidenceof lysosomal storage diseases (LSD) is 1 in 10,000 births world wide,and in 65% of cases, there is significant central nervous system (CNS)involvement. Proteins deficient in these disorders, when deliveredintravenously, do not cross the blood-brain barrier, or, when delivereddirectly to the brain, are not widely distributed. Thus, therapies forthe CNS deficits need to be developed.

SUMMARY

The present inventors have discovered peptides that function to targetagents, such as viral vectors, to vascular endothelial cells of thecentral nervous system. The present disclosure describes a method toutilize these novel peptides to redirect, for example, viral capsids tothe cell type of interest. In this instance, endothelial cells liningbrain blood vessels are targeted by the identified peptides. Vectorsharboring capsid proteins modified to include such peptides can be usedto provide therapeutic agents to the central nervous system (e.g., thebrain).

As used herein, the term “targets” means that the capsid protein of avirus, such as an adeno-associated virus (AAV), preferentially binds toone type of tissue (e.g., liver tissue) over another type of tissue(e.g., brain tissue), and/or binds to a tissue in a certain state (e.g.,wildtype or diseased). In certain embodiments, the genetically modifiedcapsid protein may “target” brain vascular epithelia tissue by bindingat level of 10% to 1000% higher than a comparable, unmodified capsidprotein. For example, an AAV having a genetically-modified capsidprotein may bind to brain vascular epithelia tissue at a level 50% to100% greater than an unmodified AAV virus. In certain embodiments, thenucleic acids encoding the capsid proteins of a virus are modified suchthat the viral capsids preferentially bind to brain vascular endotheliumin a mammal suffering from lysosomal storage disease, or, usingdifferent sequences, to wildtype brain vascular endothelium in brain ofthe same species.

The present invention provides a modified adeno-associated virus (AAV)capsid protein containing a targeting peptide, wherein the targetingpeptide is from 3 to 10 amino acids in length and wherein the targetingpeptide targets an AAV to brain vascular endothelium. In certainembodiments, the targeting peptide is 3, 4, 5, 6 or 7 amino acids inlength. In certain embodiments, the AAV is AAV2, although the tropism ismodified so it would follow that such modifications would change thetropism of any AAV.

In certain embodiments, the targeting peptide targets wildtype brainvascular endothelium. In certain embodiments, the targeting peptide isPXXPS (SEQ ID NO:1), SPXXP (SEQ ID NO:2), TLH (SEQ ID NO:3), or QSXY(SEQ ID NO:4), as expressed in an amino to carboxy orientation or in acarboxy to amino orientation. In certain embodiments, the targetingpeptide is PYFPSLS (SEQ ID NO:5), YAPLTPS (SEQ ID NO:6), PLSPSAY (SEQ IDNO:7), DSPAHPS (SEQ ID NO:8), GTPTHPS (SEQ ID NO:9), PDAPSNH (SEQ IDNO:10), TEPHWPS (SEQ ID NO:11), SPPLPPK (SEQ ID NO:12), SPKPPPG (SEQ IDNO:13), NWSPWDP (SEQ ID NO:14), DSPAHPS (SEQ ID NO:15), GWTLHNK (SEQ IDNO:16), KIPPTLH (SEQ ID NO:17), ISQTLHG (SEQ ID NO:18), QSFYILT (SEQ IDNO:19), or TTQSEYG (SEQ ID NO:20), as expressed in an amino to carboxyorientation or in a carboxy to amino orientation. It should be notedthat the orientation of the sequence is not important. For example, thepeptide may be oriented from the amino-terminal end to carboxy-terminalend of the peptide to be TTQSEYG (SEQ ID NO:20) or may be from theamino-terminal end to carboxy-terminal end of the peptide to be GYESQTT(SEQ ID NO:42).

In certain embodiments, the targeting peptide targets a diseased brainvascular endothelium. In certain embodiments, the targeting peptidetargets brain vascular endothelium in a subject that has a lysosomalstorage disease. In certain embodiments, the targeting peptide targets amucopolysaccharide (MPS) VII brain vascular endothelium. In certainembodiments, the targeting peptide is LXSS (SEQ ID NO:21), PFXG (SEQ IDNO:22), or SIXA (SEQ ID NO:23), as expressed in an amino to carboxyorientation or in a carboxy to amino orientation. In certainembodiments, the targeting peptide is MLVSSPA (SEQ ID NO:24), LPSSLQK(SEQ ID NO:25), PPLLKSS (SEQ ID NO:26), PXKLDSS (SEQ ID NO:27), AWTLASS(SEQ ID NO:28), WPFYGTP (SEQ ID NO:29), GTFPFLG (SEQ ID NO:30), GQVPFMG(SEQ ID NO:31), ANFSILA (SEQ ID NO:32), GSIWAPA (SEQ ID NO:33), orSIAASFS (SEQ ID NO:34), as expressed in an amino to carboxy orientationor in a carboxy to amino orientation.

In certain embodiments, targeting peptide targets TPP1-deficient-brainvascular endothelium. In certain embodiments, the targeting peptide isGMNAFRA (SEQ ID NO:41), as expressed in an amino to carboxy orientationor in a carboxy to amino orientation.

The present invention provides a nucleic acid sequence encoding amodified capsid described hereinabove.

The present invention provides an AAV virus containing the capsidprotein modified genetically to encode the peptides describedhereinabove.

The present invention provides a viral vector comprising a nucleic acidencoding the capsid protein as described hereinabove. In certainembodiments, the viral vector further contains a nucleic acid sequenceencoding a nucleic acid of interest. In certain embodiments, the nucleicacid of interest is a therapeutic agent. In certain embodiments, thetherapeutic agent is an enzyme or an RNAi molecule (e.g., siRNA, shRNAor miRNA molecules). In certain embodiments, the therapeutic agent isβ-glucuronidase or tripeptidyl protease.

The present invention provides a cell containing the viral vectordescribed hereinabove.

The present invention provides a cell transduced by the viral vectordescribed hereinabove. In certain embodiments, the cell is a mammaliancell. In certain embodiments, the cell is a human cell. In otherembodiments, the cell is a non-human cell. In certain embodiments, thecell is in vitro, and in other embodiments, the cell is in vivo. Incertain embodiments, the cell is an endothelial cell. In certainembodiments, the cell is a vascular endothelial cell.

The present invention provides a method of treating the brain disease ina mammal by administering the viral vector described hereinabove or thecell described hereinabove to the mammal. In certain embodiments, themammal is human. In certain embodiments, the disease is a lysosomalstorage disease (LSD), such as infantile or late infantile ceroidlipofuscinoses, neuronopathic Gaucher, Juvenile Batten, Fabry, MLD,Sanfilippo A, Hunter, Krabbe, Morquio, Pompe, Niemann-Pick C, Tay-Sachs,Hurler (MPS-I H), Sanfilippo B, Maroteaux-Lamy, Niemann-Pick A,Cystinosis, Hurler-Scheie (MPS-I H/S), Sly Syndrome (MPS VII), Scheie(MPS-I S), Infantile Batten, GM1 Gangliosidosis, Mucolipidosis typeII/III, or Sandhoff disease. In certain embodiments, the disease is aneurodegenerative disease, such as Huntington's disease, ALS, hereditaryspastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy,Kennedy's disease, Alzheimer's disease, a polyglutamine repeat disease,or Parkinson's disease.

The present invention provides a method to deliver an agent to thecentral nervous system of a subject, by transducing vascular endothelialcells with a viral vector described hereinabove so that the transducedvascular endothelial cells express the therapeutic agent and deliver theagent to the central nervous system of the subject. In certainembodiments, the viral vector transduces vascular endothelial cells

The present invention provides a viral vector as described hereinabovefor use in medical treatment or diagnosis.

The present invention provides a use of the viral vector describedhereinabove to prepare a medicament useful for treating a lysosomalstorage disease in a mammal.

The present invention provides a cell as described hereinabove for usein medical treatment or diagnosis.

The present invention provides a use of the cell as describedhereinabove to prepare a medicament useful for treating a lysosomalstorage disease in a mammal.

The present invention provides a method for identifying peptides thattarget brain vascular endothelium by using phage display biopanning soas to identify such peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. FIG. 1A depicts a vascular cast of the human brain, and1B shows four cells of the CNS microvasculature.

FIGS. 2A-2D. In vivo phage display panning to identify peptide motifswith high affinity for cerebral vasculature. (FIG. 2A, 2B) After 5rounds of in vivo phage display panning, phage with distinct peptidemotifs were identified from wildtype (a) and MPS VII (b) mice. (FIGS.2C, 2D) Selected phage were individually injected via tail vein, andphage was recovered and tittered from cerebral vasculature of wildtype(c) (SEQ ID NOS 5-14, 16-18, 20 and 19, respectively in order ofappearance) and MPS VII (d) mice (SEQ ID NOS 29-31, 26, 34, 28, 25, 27,33, 32 and 24, respectively in order of appearance). Data presented asmean±SEM.

FIGS. 3A-3B. Peptide-modified virus exhibits selective transduction ofcerebral vasculature independent of heparin sulfate. (FIGS. 3A, 3B) Fourweeks after tail vein injection of peptide-modified virus (1.0×10¹¹genome particles/mouse), viral genomes were quantified by RT-PCR inbrain and liver of wildtype (a) and MPS VII (b) mice.

FIGS. 4A-4K. Intravenous delivery of peptide-modified virus rescuesneuropathology and CNS deficits of MPS VII mice. (FIGS. 4A-4H)Toluidine-blue stained sections (1 μm) through cerebral cortex (ctx),hippocampus (hc), striatum (str), and cerebellum (cb) of MPS VII miceinjected via tail vein with either AAV-WT or AAV-PFG expressingβ-glucuronidase. Representative images are shown. (FIG. 4I) In thecontext fear conditioning assay, MPS VII mice treated with AAV-WTcontrol virus (n=4), MPS VII mice treated with AAV-PFG (n=6), andheterozygous controls (n=6) were tested for their ability todiscriminate a harmful vs benign context (see methods). Decreases infreezing time corresponds to intact context discrimination. Datapresented as mean±SEM, *p<0.05. (FIG. 4J) Binding of AAV-PFG to cerebralvasculature requires chondroitin sulfate. Purified brain vasculaturesfrom wildtype (WT) or MPS VII (MPS) mice were pre-incubated with PBSalone, PNGase (100 U/reaction), or chondroitinase ABC (2 U/reaction).Vasculatures were then incubated with wildtype AAV or AAV-PFG (1.0×10¹¹genome particles) in 500 μl PBS. Bound viral particles were quantifiedby RT-PCR. Data presented as mean±SEM. (FIG. 4K) Binding of AAV-PFG topurified brain vasculature from MPS VII mice in the presence or absenceof 2 mg/ml chondroitin sulfate. Data presented as mean±SEM.

FIGS. 5A-5E. In vivo phage display panning in TPP1-deficient (CLN2 −/−)mice. (FIG. 5A) After 5 rounds of panning, a single peptide wasrecovered—ARFANMG (SEQ ID NO: 43). AAV modified with this peptide wastittered at 1.76×10¹² viral genomes/ml. (FIGS. 5B-5D) Immunostaining forTPP1 3 weeks after tail vein injection of modified virus (1.76×10¹¹viral genomes) into TPP1-deficient mice reveals enzyme in cerebralcortex (b), midbrain (c), and cerebellum (d). Scale bars, 50 μm. (FIG.5E) In vitro assay for TPP1 activity in several tissues following tailvein injection of peptide modified virus. Activity levels expressedrelative to heterozygous control.

DETAILED DESCRIPTION

Certain embodiments of the present disclosure provide a viral vectorcomprising a modified capsid, wherein the modified capsid comprises atleast one amino acid sequence that targets the viral vector to brainvascular endothelium.

In certain embodiments, the viral vector is an adeno associated viralvector (AAV). In certain embodiments, the AAV is AAV2.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises or consists of PXXPS (SEQ ID NO:1), SPXXP(SEQ ID NO:2), TLH (SEQ ID NO:3), QSXY (SEQ ID NO:4), LXSS (SEQ IDNO:21), PFXG (SEQ ID NO:22), or SIXA (SEQ ID NO:23), as expressed in anamino to carboxy orientation or in a carboxy to amino orientation.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises or consists of PYFPSLS (SEQ ID NO:5),YAPLTPS (SEQ ID NO:6), PLSPSAY (SEQ ID NO:7), DSPAHPS (SEQ ID NO:8),GTPTHPS (SEQ ID NO:9), PDAPSNH (SEQ ID NO:10), TEPHWPS (SEQ ID NO:11),SPPLPPK (SEQ ID NO:12), SPKPPPG (SEQ ID NO:13), NWSPWDP (SEQ ID NO:14),DSPAHPS (SEQ ID NO:15), GWTLHNK (SEQ ID NO:16), KIPPTLH (SEQ ID NO:17),ISQTLHG (SEQ ID NO:18), QSFYILT (SEQ ID NO:19), TTQSEYG (SEQ ID NO:20),MLVSSPA (SEQ ID NO:24), LPSSLQK (SEQ ID NO:25), PPLLKSS (SEQ ID NO:26),PXKLDSS (SEQ ID NO:27), AWTLASS (SEQ ID NO:28), WPFYGTP (SEQ ID NO:29),GTFPFLG (SEQ ID NO:30), GQVPFMG (SEQ ID NO:31), ANFSILA (SEQ ID NO:32),GSIWAPA (SEQ ID NO:33), or SIAASFS (SEQ ID NO:34), as expressed in anamino to carboxy orientation or in a carboxy to amino orientation.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises or consists of GMNAFRA (SEQ ID NO:41), asexpressed in an amino to carboxy orientation or in a carboxy to aminoorientation.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises at least one of SEQ ID NOs 1-4.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises at least one of SEQ ID NOs 21-23.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises at least one of SEQ ID NOs 5-20.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium comprises at least one of SEQ ID NOs 24-34.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium targets brain vascular endothelium in a subjectthat has a disease, e.g., a lysosomal storage disease.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium targets brain vascular endothelium in a subjectthat does not have a lysosomal storage disease.

In certain embodiments, the viral vector comprises a nucleic acidsequence encoding a therapeutic agent. In certain embodiments, thetherapeutic agent is β-glucuronidase.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium is at most ten amino acids in length.

In certain embodiments, the amino acid sequence that targets brainvascular endothelium is 3, 4, 5, 6 or 7 amino acids in length.

Certain embodiments of the present disclosure provide a nucleic acidsequence encoding a viral vector as described herein.

Certain embodiments of the present disclosure provide a nucleic acidsequence encoding a modified capsid as described herein. Certainembodiments of the present disclosure provide a modified capsid encodedby a nucleic acid sequence described herein.

Certain embodiments of the present disclosure provide a cell comprisinga viral vector as described herein.

Certain embodiments of the present disclosure provide a cell transducedby a viral vector as described herein.

In certain embodiments, the cell is a mammalian cell. In certainembodiments, the cell is a human cell. In certain embodiments, the cellis a non-human cell. In certain embodiments, the cell is in vitro. Incertain embodiments, the cell is in vivo. In certain embodiments, thecell is an endothelial cell. In certain embodiments, the cell is avascular endothelial cell.

Certain embodiments of the present disclosure provide a method oftreating a disease in a mammal comprising administering a viral vectoror the cell as described herein to the mammal.

In certain embodiments, the mammal is human.

In certain embodiments, the disease is a lysosomal storage disease(LSD).

In certain embodiments, the LSD is infantile or late infantile ceroidlipofuscinoses, Gaucher, Juvenile Batten, Fabry, MLD, Sanfilippo A, LateInfantile Batten, Hunter, Krabbe, Morquio, Pompe, Niemann-Pick C,Tay-Sachs, Hurler (MPS-I H), Sanfilippo B, Maroteaux-Lamy, Niemann-PickA, Cystinosis, Hurler-Scheie (MPS-I H/S), Sly Syndrome (MPS VII), Scheie(MPS-I S), Infantile Batten, GM1 Gangliosidosis, Mucolipidosis typeII/III, or Sandhoff disease.

In certain embodiments, the disease is a neurodegenerative disease.

In certain embodiments, the neurodegenerative disease is Huntington'sdisease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis,spinal muscular atrophy, Kennedy's disease, Alzheimer's disease, apolyglutamine repeat disease, or Parkinson's disease.

Certain embodiments of the present disclosure provide a method todeliver an agent to the central nervous system of a subject, comprisingtransducing vascular endothelial cells with a viral vector describedherein so that the transduced vascular endothelial cells express thetherapeutic agent and deliver the agent to the central nervous system ofthe subject.

In certain embodiments, the viral vector transduces vascular endothelialcells.

Certain embodiments of the present disclosure provide a viral vector orcell as described herein for use in medical treatment or diagnosis.

Certain embodiments of the present disclosure provide a use of a viralvector or cell as described herein to prepare a medicament useful fortreating a disease, e.g., a lysosomal storage disease, in a mammal.

Certain embodiments of the present disclosure provide a method foridentifying peptides that target brain vascular endothelium comprisingusing phage display biopanning so as to identify such peptides.

The vector may further comprise a lysosomal enzyme, a secreted protein,a nuclear protein, or a cytoplasmic protein. As used herein, the term“secreted protein” includes any secreted protein, whether naturallysecreted or modified to contain a signal sequence so that it can besecreted. For example, the secreted protein could be β-glucuronidase,pepstatin insensitive protease, palmitoyl protein thioesterase.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. Generally, “operablylinked” means that the DNA sequences being linked are contiguous.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. Additionally, multiple copies of the nucleicacid encoding enzymes may be linked together in the expression vector.Such multiple nucleic acids may be separated by linkers.

The vector may be an adeno-associated virus (AAV) vector, an adenoviralvector, a retrovirus, or a lentivirus vector based on humanimmunodeficiency virus or feline immunodeficiency virus. Examples ofsuch AAVs are found in Davidson et al., PNAS (2000) 97:3428-3432. TheAAV and lentiviruses can confer lasting expression while the adenoviruscan provide transient expression.

The present disclosure also provides a mammalian cell containing avector described herein. The cell may be human, and may be from brain,spleen, kidney, lung, heart, or liver. The cell type may be a stem orprogenitor cell population.

The present disclosure provides a method of treating a disease such as agenetic disease or cancer in a mammal by administering a polynucleotide,polypeptide, expression vector, or cell described herein. The geneticdisease or cancer may be a lysosomal storage disease (LSD) such asinfantile or late infantile ceroid lipofuscinoses, Gaucher, JuvenileBatten, Fabry, MLD, Sanfilippo A, Late Infantile Batten, Hunter, Krabbe,Morquio, Pompe, Niemann-Pick C, Tay-Sachs, Hurler (MPS-I H), SanfilippoB, Maroteaux-Lamy, Niemann-Pick A, Cystinosis, Hurler-Scheie (MPS-IH/S), Sly Syndrome (MPS VII), Scheie (MPS-I S), Infantile Batten, GM1Gangliosidosis, Mucolipidosis type II/III, or Sandhoff disease.

The genetic disease may be a neurodegenerative disease, such asHuntington's disease, ALS, hereditary spastic hemiplegia, primarylateral sclerosis, spinal muscular atrophy, Kennedy's disease,Alzheimer's disease, a polyglutamine repeat disease, or focal exposuresuch as Parkinson's disease.

Certain aspects of the disclosure relate to polynucleotides,polypeptides, vectors, and genetically engineered cells (modified invivo), and the use of them. In particular, the disclosure relates to amethod for gene or protein therapy that is capable of both systemicdelivery of a therapeutically effective dose of the therapeutic agent.

According to one aspect, a cell expression system for expressing atherapeutic agent in a mammalian recipient is provided. The expressionsystem (also referred to herein as a “genetically modified cell”)comprises a cell and an expression vector for expressing the therapeuticagent. Expression vectors include, but are not limited to, viruses,plasmids, and other vehicles for delivering heterologous geneticmaterial to cells. Accordingly, the term “expression vector” as usedherein refers to a vehicle for delivering heterologous genetic materialto a cell. In particular, the expression vector is a recombinantadenoviral, adeno-associated virus, or lentivirus or retrovirus vector.

The expression vector further includes a promoter for controllingtranscription of the heterologous gene. The promoter may be an induciblepromoter (described below). The expression system is suitable foradministration to the mammalian recipient. The expression system maycomprise a plurality of non-immortalized genetically modified cells,each cell containing at least one recombinant gene encoding at least onetherapeutic agent.

The cell expression system can be formed in vivo. According to yetanother aspect, a method for treating a mammalian recipient in vivo isprovided. The method includes introducing an expression vector forexpressing a heterologous gene product into a cell of the patient insitu, such as via intravenous administration. To form the expressionsystem in vivo, an expression vector for expressing the therapeuticagent is introduced in vivo into the mammalian recipient i.v., where thevector migrates via the vasculature to the brain.

According to yet another aspect, a method for treating a mammalianrecipient in vivo is provided. The method includes introducing thetarget protein into the patient in vivo.

The expression vector for expressing the heterologous gene may includean inducible promoter for controlling transcription of the heterologousgene product. Accordingly, delivery of the therapeutic agent in situ iscontrolled by exposing the cell in situ to conditions, which inducetranscription of the heterologous gene.

The mammalian recipient may have a condition that is amenable to genereplacement therapy. As used herein, “gene replacement therapy” refersto administration to the recipient of exogenous genetic materialencoding a therapeutic agent and subsequent expression of theadministered genetic material in situ. Thus, the phrase “conditionamenable to gene replacement therapy” embraces conditions such asgenetic diseases (i.e., a disease condition that is attributable to oneor more gene defects), acquired pathologies (i.e., a pathologicalcondition which is not attributable to an inborn defect), cancers andprophylactic processes (i.e., prevention of a disease or of an undesiredmedical condition). Accordingly, as used herein, the term “therapeuticagent” refers to any agent or material, which has a beneficial effect onthe mammalian recipient. Thus, “therapeutic agent” embraces boththerapeutic and prophylactic molecules having nucleic acid or proteincomponents.

According to one embodiment, the mammalian recipient has a geneticdisease and the exogenous genetic material comprises a heterologous geneencoding a therapeutic agent for treating the disease. In yet anotherembodiment, the mammalian recipient has an acquired pathology and theexogenous genetic material comprises a heterologous gene encoding atherapeutic agent for treating the pathology. According to anotherembodiment, the patient has a cancer and the exogenous genetic materialcomprises a heterologous gene encoding an anti-neoplastic agent. In yetanother embodiment the patient has an undesired medical condition andthe exogenous genetic material comprises a heterologous gene encoding atherapeutic agent for treating the condition.

According to yet another embodiment, a pharmaceutical composition isdisclosed. The pharmaceutical composition comprises a plurality of theabove-described genetically modified cells or polypeptides and apharmaceutically acceptable carrier. The pharmaceutical composition maybe for treating a condition amenable to gene replacement therapy and theexogenous genetic material comprises a heterologous gene encoding atherapeutic agent for treating the condition. The pharmaceuticalcomposition may contain an amount of genetically modified cells orpolypeptides sufficient to deliver a therapeutically effective dose ofthe therapeutic agent to the patient. Exemplary conditions amenable togene replacement therapy are described below.

According to another aspect, a method for forming the above-describedpharmaceutical composition is provided. The method includes introducingan expression vector for expressing a heterologous gene product into acell to form a genetically modified cell and placing the geneticallymodified cell in a pharmaceutically acceptable carrier.

These and other aspects, as well as various advantages and utilitieswill be more apparent with reference to the detailed description and tothe accompanying Figures.

As used herein, the term “lysosomal enzyme,” a “secreted protein,” a“nuclear protein,” or a “cytoplasmic protein” include variants orbiologically active or inactive fragments of these polypeptides. A“variant” of one of the polypeptides is a polypeptide that is notcompletely identical to a native protein. Such variant protein can beobtained by altering the amino acid sequence by insertion, deletion orsubstitution of one or more amino acid. The amino acid sequence of theprotein is modified, for example by substitution, to create apolypeptide having substantially the same or improved qualities ascompared to the native polypeptide. The substitution may be a conservedsubstitution. A “conserved substitution” is a substitution of an aminoacid with another amino acid having a similar side chain. A conservedsubstitution would be a substitution with an amino acid that makes thesmallest change possible in the charge of the amino acid or size of theside chain of the amino acid (alternatively, in the size, charge or kindof chemical group within the side chain) such that the overall peptideretains its spacial conformation but has altered biological activity.For example, common conserved changes might be Asp to Glu, Asn or Gln;His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr orGly. Alanine is commonly used to substitute for other amino acids. The20 essential amino acids can be grouped as follows: alanine, valine,leucine, isoleucine, proline, phenylalanine, tryptophan and methioninehaving nonpolar side chains; glycine, serine, threonine, cystine,tyrosine, asparagine and glutamine having uncharged polar side chains;aspartate and glutamate having acidic side chains; and lysine, arginine,and histidine having basic side chains.

The amino acid changes are achieved by changing the codons of thecorresponding nucleic acid sequence. It is known that such polypeptidescan be obtained based on substituting certain amino acids for otheramino acids in the polypeptide structure in order to modify or improvebiological activity. For example, through substitution of alternativeamino acids, small conformational changes may be conferred upon apolypeptide that results in increased activity. Alternatively, aminoacid substitutions in certain polypeptides may be used to provideresidues, which may then be linked to other molecules to providepeptide-molecule conjugates which, retain sufficient properties of thestarting polypeptide to be useful for other purposes.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, wherein it is foundthat certain amino acids may be substituted for other amino acids havingsimilar hydropathic indices and still retain a similar biologicalactivity. Alternatively, substitution of like amino acids may be made onthe basis of hydrophilicity, particularly where the biological functiondesired in the polypeptide to be generated in intended for use inimmunological embodiments. The greatest local average hydrophilicity ofa “protein”, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity. Accordingly, it is noted thatsubstitutions can be made based on the hydrophilicity assigned to eachamino acid.

In using either the hydrophilicity index or hydropathic index, whichassigns values to each amino acid, it is preferred to conductsubstitutions of amino acids where these values are ±2, with ±1 beingparticularly preferred, and those with in ±0.5 being the most preferredsubstitutions.

The variant protein has at least 50%, at least about 80%, or even atleast about 90% but less than 100%, contiguous amino acid sequencehomology or identity to the amino acid sequence of a correspondingnative protein.

The amino acid sequence of the variant polypeptide correspondsessentially to the native polypeptide's amino acid sequence. As usedherein “correspond essentially to” refers to a polypeptide sequence thatwill elicit a biological response substantially the same as the responsegenerated by the native protein. Such a response may be at least 60% ofthe level generated by the native protein, and may even be at least 80%of the level generated by native protein.

A variant may include amino acid residues not present in thecorresponding native protein or deletions relative to the correspondingnative protein. A variant may also be a truncated “fragment” as comparedto the corresponding native protein, i.e., only a portion of afull-length protein. Protein variants also include peptides having atleast one D-amino acid.

The variant protein may be expressed from an isolated DNA sequenceencoding the variant protein. “Recombinant” is defined as a peptide ornucleic acid produced by the processes of genetic engineering. It shouldbe noted that it is well-known in the art that, due to the redundancy inthe genetic code, individual nucleotides can be readily exchanged in acodon, and still result in an identical amino acid sequence. The terms“protein,” “peptide” and “polypeptide” are used interchangeably herein.

The present disclosure provides methods of treating a disease in amammal by administering an expression vector to a cell or patient. Forthe gene therapy methods, a person having ordinary skill in the art ofmolecular biology and gene therapy would be able to determine, withoutundue experimentation, the appropriate dosages and routes ofadministration of the expression vector used in the novel methods of thepresent disclosure.

According to one embodiment, the cells are transformed or otherwisegenetically modified in vivo. The cells from the mammalian recipient aretransformed (i.e., transduced or transfected) in vivo with a vectorcontaining exogenous genetic material for expressing a heterologous(e.g., recombinant) gene encoding a therapeutic agent and thetherapeutic agent is delivered in situ.

As used herein, “exogenous genetic material” refers to a nucleic acid oran oligonucleotide, either natural or synthetic, that is not naturallyfound in the cells; or if it is naturally found in the cells, it is nottranscribed or expressed at biologically significant levels by thecells. Thus, “exogenous genetic material” includes, for example, anon-naturally occurring nucleic acid that can be transcribed intoanti-sense RNA, as well as a “heterologous gene” (i.e., a gene encodinga protein which is not expressed or is expressed at biologicallyinsignificant levels in a naturally-occurring cell of the same type).

In the certain embodiments, the mammalian recipient has a condition thatis amenable to gene replacement therapy. As used herein, “genereplacement therapy” refers to administration to the recipient ofexogenous genetic material encoding a therapeutic agent and subsequentexpression of the administered genetic material in situ. Thus, thephrase “condition amenable to gene replacement therapy” embracesconditions such as genetic diseases (i.e., a disease condition that isattributable to one or more gene defects), acquired pathologies (i.e., apathological condition which is not attributable to an inborn defect),cancers and prophylactic processes (i.e., prevention of a disease or ofan undesired medical condition). Accordingly, as used herein, the term“therapeutic agent” refers to any agent or material, which has abeneficial effect on the mammalian recipient. Thus, “therapeutic agent”embraces both therapeutic and prophylactic molecules having nucleic acid(e.g., antisense RNA) and/or protein components.

A number of lysosomal storage diseases are known (for exampleNeimann-Pick disease, Sly syndrome, Gaucher Disease). Other examples oflysosomal storage diseases are provided in Table 1. Therapeutic agentseffective against these diseases are also known, since it is theprotein/enzyme known to be deficient in these disorders.

TABLE 1 List of putative target diseases for gene therapies. DiseaseGaucher Juvenile Batten Fabry MLD Sanfilippo A Late Infantile BattenHunter Krabbe Morquio Pompe Niemann-Pick C Tay-Sachs Hurler (MPS-I H)Sanfilippo B Maroteaux-Lamy Niemann-Pick A Cystinosis Hurler-Scheie(MPS-I H/S) Sly Syndrome (MPS VII) Scheie (MPS-I S) Infantile Batten GM1Gangliosidosis Mucolipidosis type II/III Sandhoff other

As used herein, “acquired pathology” refers to a disease or syndromemanifested by an abnormal physiological, biochemical, cellular,structural, or molecular biological state. Exemplary acquiredpathologies, are provided in Table 2. Therapeutic agents effectiveagainst these diseases are also given.

TABLE II Potential Gene Therapies for Motor Neuron Diseases and otherneurodegenerative diseases. Candidates for Neuronal or Candidates forGene Downstream Progenitor Cell Disease Replacement² Effectors³Replacement⁴ ALS No Yes Yes Hereditary Spastin, paraplegin Yes Yesspastic hemiplegia Primary lateral No Yes Yes sclerosis⁵ Spinal Survivalmotor neuron Yes Yes muscular gene, neuronal atrophy apoptosisinhibiting factor Kennedy's Androgen-receptor Yes Yes disease elementAlzheimer's Yes Yes disease Polyglutamine Yes Yes Repeat Diseases ²Basedon current literature. ³Based on current literature, includes calbindin,trophic factors, bcl-2, neurofilaments, and pharmacologic agents. ⁴Mayinclude cell- or cell- and gene-based therapies. ⁵A sporadicdegeneration of corticospinal neurons, 1/100^(th) as common as ALS, withno known genetic links.

Alternatively, the condition amenable to gene replacement therapy is aprophylactic process, i.e., a process for preventing disease or anundesired medical condition. Thus, the instant disclosure embraces acell expression system for delivering a therapeutic agent that has aprophylactic function (i.e., a prophylactic agent) to the mammalianrecipient.

In summary, the term “therapeutic agent” includes, but is not limitedto, the agents listed in the Tables above, as well as their functionalequivalents. As used herein, the term “functional equivalent” refers toa molecule (e.g., a peptide or protein) that has the same or an improvedbeneficial effect on the mammalian recipient as the therapeutic agent ofwhich is it deemed a functional equivalent. As will be appreciated byone of ordinary skill in the art, a functionally equivalent proteins canbe produced by recombinant techniques, e.g., by expressing a“functionally equivalent DNA.” As used herein, the term “functionallyequivalent DNA” refers to a non-naturally occurring DNA, which encodes atherapeutic agent. For example, many, if not all, of the agentsdisclosed in Tables 1-2 have known amino acid sequences, which areencoded by naturally occurring nucleic acids. However, due to thedegeneracy of the genetic code, more than one nucleic acid can encodethe same therapeutic agent. Accordingly, the instant disclosure embracestherapeutic agents encoded by naturally-occurring DNAs, as well as bynon-naturally-occurring DNAs, which encode the same protein as, encodedby the naturally-occurring DNA.

The above-disclosed therapeutic agents and conditions amenable to genereplacement therapy are merely illustrative and are not intended tolimit the scope of the instant disclosure. The selection of a suitabletherapeutic agent for treating a known condition is deemed to be withinthe scope of one of ordinary skill of the art without undueexperimentation.

Screening Methods

The present disclosure provides methods to screen for and identify aminoacid sequences that target, e.g., specifically target, a specific area,such as the vasculature of the central nervous system. This method canbe used to identify targeting sequences that are specific for specificdisease states. In other words, targeting sequences may be identifiedand used in the treatment of specific diseases.

AAV Vectors

Adeno associated virus (AAV) is a small (20 nm), nonpathogenic virusthat is useful in treating human brain diseases, such as Parkinson'sdisease and recessive genetic diseases. A construct is generated thatsurrounds a promoter linked to a beta-glucuronidase gene with AAV ITRsequences.

In one embodiment, a viral vector of the disclosure is an AAV vector. An“AAV” vector refers to an adeno-associated virus, and may be used torefer to the naturally occurring wild-type virus itself or derivativesthereof. The term covers all subtypes, serotypes and pseudotypes, andboth naturally occurring and recombinant forms, except where requiredotherwise. As used herein, the term “serotype” refers to an AAV which isidentified by and distinguished from other AAVs based on capsid proteinreactivity with defined antisera, e.g., there are eight known serotypesof primate AAVs, AAV-1 to AAV-8. For example, serotype AAV-2 is used torefer to an AAV which contains capsid proteins encoded from the cap geneof AAV-2 and a genome containing 5′ and 3′ ITR sequences from the sameAAV-2 serotype. Pseudotyped AAV refers to an AAV that contains capsidproteins from one serotype and a viral genome including 5′-3′ ITRs of asecond serotype. Pseudotyped rAAV would be expected to have cell surfacebinding properties of the capsid serotype and genetic propertiesconsistent with the ITR serotype. Pseudotyped rAAV are produced usingstandard techniques described in the art. As used herein, for example,rAAV1 may be used to refer an AAV having both capsid proteins and 5′-3′ITRs from the same serotype or it may refer to an AAV having capsidproteins from serotype 1 and 5′-3′ ITRs from a different AAV serotype,e.g., AAV serotype 2. For each example illustrated herein thedescription of the vector design and production describes the serotypeof the capsid and 5′-3′ ITR sequences. The abbreviation “rAAV” refers torecombinant adeno-associated virus, also referred to as a recombinantAAV vector (or “rAAV vector”).

An “AAV virus” or “AAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein (preferably by all of thecapsid proteins of a wild-type AAV) and an encapsidated polynucleotide.If the particle comprises heterologous polynucleotide (i.e., apolynucleotide other than a wild-type AAV genome such as a transgene tobe delivered to a mammalian cell), it is typically referred to as“rAAV”.

In one embodiment, the AAV expression vectors are constructed usingknown techniques to at least provide as operatively linked components inthe direction of transcription, control elements including atranscriptional initiation region, the DNA of interest and atranscriptional termination region. The control elements are selected tobe functional in a mammalian cell. The resulting construct whichcontains the operatively linked components is flanked (5′ and 3′) withfunctional AAV ITR sequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision and rescue from, andintegration of a nucleotide sequence interposed between two flankingITRs into a mammalian cell genome.

The nucleotide sequences of AAV ITR regions are known. See for exampleKotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I.“Parvoviridae and their Replication” in Fundamental Virology, 2ndEdition, (B. N. Fields and D. M. Knipe, eds.). As used herein, an “AAVITR” need not have the wild-type nucleotide sequence depicted, but maybe altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flanka selected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the heterologous sequence into the recipient cell genomewhen AAV Rep gene products are present in the cell.

In one embodiment, AAV ITRs can be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAVX7, etc. Furthermore, 5′ and 3′ ITRs which flank a selectednucleotide sequence in an AAV expression vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector, and to allowintegration of the DNA molecule into the recipient cell genome when AAVRep gene products are present in the cell.

In one embodiment, AAV capsids can be derived from any of several AAVserotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV6, or AAV8, and the AAV ITRS are derived form AAV serotype 2.Suitable DNA molecules for use in AAV vectors will be less than about 5kilobases (kb), less than about 4.5 kb, less than about 4 kb, less thanabout 3.5 kb, less than about 3 kb, less than about 2.5 kb in size andare known in the art.

In some embodiments of the disclosure the DNA molecules for use in theAAV vectors will contain one or more copies of a single siRNA sequence.As used herein the term multiple copies of an siRNA sequences means atleast 2 copies, at least 3 copies, at least 4 copies, at least 5 copies,at least 6 copies, at least 7 copies, at least 8 copies, at least 9copies, and at least 10 copies. In some embodiments the DNA moleculesfor use in the AAV vectors will contain multiple siRNA sequences. Asused herein the term multiple siRNA sequences means at least 2 siRNAsequences, at least 3 siRNA sequences, at least 4 siRNA sequences, atleast 5 siRNA sequences, at least 6 siRNA sequences, at least 7 siRNAsequences, at least 8 siRNA sequences, at least 9 siRNA sequences, andat least 10 siRNA sequences. In some embodiments suitable DNA vectors ofthe disclosure will contain a sequence encoding the siRNA molecule ofthe disclosure and a stuffer fragment. Suitable stuffer fragments of thedisclosure include sequences known in the art including withoutlimitation sequences which do not encode an expressed protein molecule;sequences which encode a normal cellular protein which would not havedeleterious effect on the cell types in which it was expressed; andsequences which would not themselves encode a functional siRNA duplexmolecule.

In one embodiment, suitable DNA molecules for use in AAV vectors will beless than about 5 kilobases (kb) in size and will include, for example,a stuffer sequence and a sequence encoding a siRNA molecule of thedisclosure. For example, in order to prevent any packaging of AAVgenomic sequences containing the rep and cap genes, a plasmid containingthe rep and cap DNA fragment may be modified by the inclusion of astuffer fragment as is known in the art into the AAV genome which causesthe DNA to exceed the length for optimal packaging. Thus, the helperfragment is not packaged into AAV virions. This is a safety feature,ensuring that only a recombinant AAV vector genome that does not exceedoptimal packaging size is packaged into virions. An AAV helper fragmentthat incorporates a stuffer sequence can exceed the wild-type genomelength of 4.6 kb, and lengths above 105% of the wild-type will generallynot be packaged. The stuffer fragment can be derived from, for example,such non-viral sources as the Lac-Z or beta-galactosidase gene.

In one embodiment, the selected nucleotide sequence is operably linkedto control elements that direct the transcription or expression thereofin the subject in vivo. Such control elements can comprise controlsequences normally associated with the selected gene. Alternatively,heterologous control sequences can be employed. Useful heterologouscontrol sequences generally include those derived from sequencesencoding mammalian or viral genes. Examples include, but are not limitedto, the SV40 early promoter, mouse mammary tumor virus LTR promoter;adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, polII promoters, pol III promoters, synthetic promoters, hybrid promoters,and the like. In addition, sequences derived from nonviral genes, suchas the murine metallothionein gene, will also find use herein. Suchpromoter sequences are commercially available from, e.g., Stratagene(San Diego, Calif.).

In one embodiment, both heterologous promoters and other controlelements, such as CNS-specific and inducible promoters, enhancers andthe like, will be of particular use. Examples of heterologous promotersinclude the CMB promoter. Examples of CNS-specific promoters includethose isolated from the genes from myelin basic protein (MBP), glialfibrillary acid protein (GFAP), and neuron specific enolase (NSE).Examples of inducible promoters include DNA responsive elements forecdysone, tetracycline, hypoxia and aufin.

In one embodiment, the AAV expression vector which harbors the DNAmolecule of interest bounded by AAV ITRs, can be constructed by directlyinserting the selected sequence(s) into an AAV genome which has had themajor AAV open reading frames (“ORFs”) excised therefrom. Other portionsof the AAV genome can also be deleted, so long as a sufficient portionof the ITRs remain to allow for replication and packaging functions.Such constructs can be designed using techniques well known in the art.

Alternatively, AAV ITRs can be excised from the viral genome or from anAAV vector containing the same and fused 5′ and 3′ of a selected nucleicacid construct that is present in another vector using standard ligationtechniques. For example, ligations can be accomplished in 20 mM Tris-ClpH 7.5, 10 mM MgCl₂, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, andeither 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for“sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligaseat 14° C. (for “blunt end” ligation). Intermolecular “sticky end”ligations are usually performed at 30-100 μg/ml total DNA concentrations(5-100 nM total end concentration). AAV vectors which contain ITRs. Inparticular, several AAV vectors are described therein which areavailable from the American Type Culture Collection (“ATCC”) underAccession Numbers 53222, 53223, 53224, 53225 and 53226.

Additionally, chimeric genes can be produced synthetically to includeAAV ITR sequences arranged 5′ and 3′ of one or more selected nucleicacid sequences. Preferred codons for expression of the chimeric genesequence in mammalian CNS cells can be used. The complete chimericsequence is assembled from overlapping oligonucleotides prepared bystandard methods.

In order to produce rAAV virions, an AAV expression vector is introducedinto a suitable host cell using known techniques, such as bytransfection. A number of transfection techniques are generally known inthe art. See, e.g., Sambrook et al. (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York.Particularly suitable transfection methods include calcium phosphateco-precipitation, direct micro-injection into cultured cells,electroporation, liposome mediated gene transfer, lipid-mediatedtransduction, and nucleic acid delivery using high-velocitymicroprojectiles.

In one embodiment, suitable host cells for producing rAAV virionsinclude microorganisms, yeast cells, insect cells, and mammalian cells,that can be, or have been, used as recipients of a heterologous DNAmolecule. The term includes the progeny of the original cell which hasbeen transfected. Thus, a “host cell” as used herein generally refers toa cell which has been transfected with an exogenous DNA sequence. Cellsfrom the stable human cell line, 293 (readily available through, e.g.,the American Type Culture Collection under Accession Number ATCCCRL1573) can be used in the practice of the present disclosure.Particularly, the human cell line 293 is a human embryonic kidney cellline that has been transformed with adenovirus type-5 DNA fragments, andexpresses the adenoviral E1a and E1b genes. The 293 cell line is readilytransfected, and provides a particularly convenient platform in which toproduce rAAV virions.

In one embodiment, host cells containing the above-described AAVexpression vectors are rendered capable of providing AAV helperfunctions in order to replicate and encapsidate the nucleotide sequencesflanked by the AAV ITRs to produce rAAV virions. AAV helper functionsare generally AAV-derived coding sequences which can be expressed toprovide AAV gene products that, in turn, function in trans forproductive AAV replication. AAV helper functions are used herein tocomplement necessary AAV functions that are missing from the AAVexpression vectors. Thus, AAV helper functions include one, or both ofthe major AAV ORFs, namely the rep and cap coding regions, or functionalhomologues thereof.

The Rep expression products have been shown to possess many functions,including, among others: recognition, binding and nicking of the AAVorigin of DNA replication; DNA helicase activity; and modulation oftranscription from AAV (or other heterologous) promoters. The Capexpression products supply necessary packaging functions. AAV helperfunctions are used herein to complement AAV functions in trans that aremissing from AAV vectors.

The term “AAV helper construct” refers generally to a nucleic acidmolecule that includes nucleotide sequences providing AAV functionsdeleted from an AAV vector which is to be used to produce a transducingvector for delivery of a nucleotide sequence of interest. AAV helperconstructs are commonly used to provide transient expression of AAV repand/or cap genes to complement missing AAV functions that are necessaryfor lytic AAV replication; however, helper constructs lack AAV ITRs andcan neither replicate nor package themselves. AAV helper constructs canbe in the form of a plasmid, phage, transposon, cosmid, virus, orvirion. A number of AAV helper constructs have been described, such asthe commonly used plasmids pAAV/Ad and pIM29+45 which encode both Repand Cap expression products. A number of other vectors have beendescribed which encode Rep and/or Cap expression products.

By “AAV rep coding region” is meant the art-recognized region of the AAVgenome which encodes the replication proteins Rep 78, Rep 68, Rep 52 andRep 40. These Rep expression products have been shown to possess manyfunctions, including recognition, binding and nicking of the AAV originof DNA replication, DNA helicase activity and modulation oftranscription from AAV (or other heterologous) promoters. The Repexpression products are collectively required for replicating the AAVgenome. Suitable homologues of the AAV rep coding region include thehuman herpesvirus 6 (HHV-6) rep gene which is also known to mediateAAV-2 DNA replication.

By “AAV cap coding region” is meant the art-recognized region of the AAVgenome which encodes the capsid proteins VP1, VP2, and VP3, orfunctional homologues thereof. These Cap expression products supply thepackaging functions which are collectively required for packaging theviral genome.

In one embodiment, AAV helper functions are introduced into the hostcell by transfecting the host cell with an AAV helper construct eitherprior to, or concurrently with, the transfection of the AAV expressionvector. AAV helper constructs are thus used to provide at leasttransient expression of AAV rep and/or cap genes to complement missingAAV functions that are necessary for productive AAV infection. AAVhelper constructs lack AAV ITRs and can neither replicate nor packagethemselves. These constructs can be in the form of a plasmid, phage,transposon, cosmid, virus, or virion. A number of AAV helper constructshave been described, such as the commonly used plasmids pAAV/Ad andpIM29+45 which encode both Rep and Cap expression products. A number ofother vectors have been described which encode Rep and/or Cap expressionproducts.

In one embodiment, both AAV expression vectors and AAV helper constructscan be constructed to contain one or more optional selectable markers.Suitable markers include genes which confer antibiotic resistance orsensitivity to, impart color to, or change the antigenic characteristicsof those cells which have been transfected with a nucleic acid constructcontaining the selectable marker when the cells are grown in anappropriate selective medium. Several selectable marker genes that areuseful in the practice of the disclosure include the hygromycin Bresistance gene (encoding Aminoglycoside phosphotranferase (APH)) thatallows selection in mammalian cells by conferring resistance to G418(available from Sigma, St. Louis, Mo.). Other suitable markers are knownto those of skill in the art.

In one embodiment, the host cell (or packaging cell) is rendered capableof providing non AAV derived functions, or “accessory functions,” inorder to produce rAAV virions. Accessory functions are non AAV derivedviral and/or cellular functions upon which AAV is dependent for itsreplication. Thus, accessory functions include at least those non AAVproteins and RNAs that are required in AAV replication, including thoseinvolved in activation of AAV gene transcription, stage specific AAVmRNA splicing, AAV DNA replication, synthesis of Cap expression productsand AAV capsid assembly. Viral-based accessory functions can be derivedfrom any of the known helper viruses.

In one embodiment, accessory functions can be introduced into and thenexpressed in host cells using methods known to those of skill in theart. Commonly, accessory functions are provided by infection of the hostcells with an unrelated helper virus. A number of suitable helperviruses are known, including adenoviruses; herpesviruses such as herpessimplex virus types 1 and 2; and vaccinia viruses. Nonviral accessoryfunctions will also find use herein, such as those provided by cellsynchronization using any of various known agents.

In one embodiment, accessory functions are provided using an accessoryfunction vector. Accessory function vectors include nucleotide sequencesthat provide one or more accessory functions. An accessory functionvector is capable of being introduced into a suitable host cell in orderto support efficient AAV virion production in the host cell. Accessoryfunction vectors can be in the form of a plasmid, phage, transposon orcosmid. Accessory vectors can also be in the form of one or morelinearized DNA or RNA fragments which, when associated with theappropriate control elements and enzymes, can be transcribed orexpressed in a host cell to provide accessory functions.

In one embodiment, nucleic acid sequences providing the accessoryfunctions can be obtained from natural sources, such as from the genomeof an adenovirus particle, or constructed using recombinant or syntheticmethods known in the art. In this regard, adenovirus-derived accessoryfunctions have been widely studied, and a number of adenovirus genesinvolved in accessory functions have been identified and partiallycharacterized. Specifically, early adenoviral gene regions E1a, E2a, E4,VAI RNA and, possibly, E1b are thought to participate in the accessoryprocess. Herpesvirus-derived accessory functions have been described.Vaccinia virus-derived accessory functions have also been described.

In one embodiment, as a consequence of the infection of the host cellwith a helper virus, or transfection of the host cell with an accessoryfunction vector, accessory functions are expressed which transactivatethe AAV helper construct to produce AAV Rep and/or Cap proteins. The Repexpression products excise the recombinant DNA (including the DNA ofinterest) from the AAV expression vector. The Rep proteins also serve toduplicate the AAV genome. The expressed Cap proteins assemble intocapsids, and the recombinant AAV genome is packaged into the capsids.Thus, productive AAV replication ensues, and the DNA is packaged intorAAV virions.

In one embodiment, following recombinant AAV replication, rAAV virionscan be purified from the host cell using a variety of conventionalpurification methods, such as CsCl gradients. Further, if infection isemployed to express the accessory functions, residual helper virus canbe inactivated, using known methods. For example, adenovirus can beinactivated by heating to temperatures of approximately 60° C. for,e.g., 20 minutes or more. This treatment effectively inactivates onlythe helper virus since AAV is extremely heat stable while the helperadenovirus is heat labile. The resulting rAAV virions are then ready foruse for DNA delivery to the CNS (e.g., cranial cavity) of the subject.

Methods of delivery of viral vectors include, but are not limited to,intra-arterial, intra-muscular, intravenous, intranasal and oral routes.Generally, rAAV virions may be introduced into cells of the CNS usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with rAAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with CNS cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, described morefully below, and the composition introduced into the subject by varioustechniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

In one embodiment, for in vivo delivery, the rAAV virions are formulatedinto pharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle or by injection into the CNS.

In one embodiment, viral vectors are delivered to the CNS viaconvection-enhanced delivery (CED) systems that can efficiently deliverviral vectors, e.g., AAV, over large regions of a subject's brain (e.g.,striatum and/or cortex). As described in detail and exemplified below,these methods are suitable for a variety of viral vectors, for instanceAAV vectors carrying therapeutic genes (e.g., siRNAs).

Any convection-enhanced delivery device may be appropriate for deliveryof viral vectors. In one embodiment, the device is an osmotic pump or aninfusion pump. Both osmotic and infusion pumps are commerciallyavailable from a variety of suppliers, for example Alzet Corporation,Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.). Typically, a viralvector is delivered via CED devices as follows. A catheter, cannula orother injection device is inserted into CNS tissue in the chosensubject. In view of the teachings herein, one of skill in the art couldreadily determine which general area of the CNS is an appropriatetarget. For example, when delivering AAV vector encoding a therapeuticgene to treat PD, the striatum is a suitable area of the brain totarget. Stereotactic maps and positioning devices are available, forexample from ASI Instruments, Warren, Mich. Positioning may also beconducted by using anatomical maps obtained by CT and/or MRI imaging ofthe subject's brain to help guide the injection device to the chosentarget. Moreover, because the methods described herein can be practicedsuch that relatively large areas of the brain take up the viral vectors,fewer infusion cannula are needed. Since surgical complications arerelated to the number of penetrations, the methods described herein alsoserve to reduce the side effects seen with conventional deliverytechniques.

In one embodiment, pharmaceutical compositions will comprise sufficientgenetic material to produce a therapeutically effective amount of thenucleic acid of interest, i.e., an amount sufficient to reduce orameliorate symptoms of the disease state in question or an amountsufficient to confer the desired benefit. The pharmaceuticalcompositions will also contain a pharmaceutically acceptable excipient.Such excipients include any pharmaceutical agent that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition, and which may be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,sorbitol, Tween80, and liquids such as water, saline, glycerol andethanol. Pharmaceutically acceptable salts can be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. A thorough discussion of pharmaceutically acceptableexcipients is available in Remington's Pharmaceutical Sciences (MackPub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Administration can be effected inone dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosagesof administration are well known to those of skill in the art and willvary with the viral vector, the composition of the therapy, the targetcells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

It should be understood that more than one transgene could be expressedby the delivered viral vector. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe CNS as described herein. Furthermore, it is also intended that theviral vectors delivered by the methods of the present disclosure becombined with other suitable compositions and therapies.

Methods for Introducing Genetic Material into Cells

The exogenous genetic material (e.g., a cDNA encoding one or moretherapeutic proteins) is introduced into the cell ex vivo or in vivo bygenetic transfer methods, such as transfection or transduction, toprovide a genetically modified cell. Various expression vectors (i.e.,vehicles for facilitating delivery of exogenous genetic material into atarget cell) are known to one of ordinary skill in the art.

As used herein, “transfection of cells” refers to the acquisition by acell of new genetic material by incorporation of added DNA. Thus,transfection refers to the insertion of nucleic acid into a cell usingphysical or chemical methods. Several transfection techniques are knownto those of ordinary skill in the art including: calcium phosphate DNAco-precipitation; DEAE-dextran; electroporation; cationicliposome-mediated transfection; and tungsten particle-faciliatedmicroparticle bombardment. Strontium phosphate DNA co-precipitation isanother possible transfection method.

In contrast, “transduction of cells” refers to the process oftransferring nucleic acid into a cell using a DNA or RNA virus. A RNAvirus (i.e., a retrovirus) for transferring a nucleic acid into a cellis referred to herein as a transducing chimeric retrovirus. Exogenousgenetic material contained within the retrovirus is incorporated intothe genome of the transduced cell. A cell that has been transduced witha chimeric DNA virus (e.g., an adenovirus carrying a cDNA encoding atherapeutic agent), will not have the exogenous genetic materialincorporated into its genome but will be capable of expressing theexogenous genetic material that is retained extrachromosomally withinthe cell.

Typically, the exogenous genetic material includes the heterologous gene(usually in the form of a cDNA comprising the exons coding for thetherapeutic protein) together with a promoter to control transcriptionof the new gene. The promoter characteristically has a specificnucleotide sequence necessary to initiate transcription. Optionally, theexogenous genetic material further includes additional sequences (i.e.,enhancers) required to obtain the desired gene transcription activity.For the purpose of this discussion an “enhancer” is simply anynon-translated DNA sequence which works contiguous with the codingsequence (in cis) to change the basal transcription level dictated bythe promoter. The exogenous genetic material may introduced into thecell genome immediately downstream from the promoter so that thepromoter and coding sequence are operatively linked so as to permittranscription of the coding sequence. A retroviral expression vector mayinclude an exogenous promoter element to control transcription of theinserted exogenous gene. Such exogenous promoters include bothconstitutive and inducible promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a gene under the control of aconstitutive promoter is expressed under all conditions of cell growth.Exemplary constitutive promoters include the promoters for the followinggenes which encode certain constitutive or “housekeeping” functions:hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase(DHFR), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvatekinase, phosphoglycerol mutase, the actin promoter, and otherconstitutive promoters known to those of skill in the art. In addition,many viral promoters function constitutively in eucaryotic cells. Theseinclude: the early and late promoters of SV40; the long terminal repeats(LTRs) of Moloney Leukemia Virus and other retroviruses; and thethymidine kinase promoter of Herpes Simplex Virus, among many others.Accordingly, any of the above-referenced constitutive promoters can beused to control transcription of a heterologous gene insert.

Genes that are under the control of inducible promoters are expressedonly or to a greater degree, in the presence of an inducing agent,(e.g., transcription under control of the metallothionein promoter isgreatly increased in presence of certain metal ions). Induciblepromoters include responsive elements (REs) which stimulatetranscription when their inducing factors are bound. For example, thereare REs for serum factors, steroid hormones, retinoic acid and cyclicAMP. Promoters containing a particular RE can be chosen in order toobtain an inducible response and in some cases, the RE itself may beattached to a different promoter, thereby conferring inducibility to therecombinant gene. Thus, by selecting the appropriate promoter(constitutive versus inducible; strong versus weak), it is possible tocontrol both the existence and level of expression of a therapeuticagent in the genetically modified cell. If the gene encoding thetherapeutic agent is under the control of an inducible promoter,delivery of the therapeutic agent in situ is triggered by exposing thegenetically modified cell in situ to conditions for permittingtranscription of the therapeutic agent, e.g., by intraperitonealinjection of specific inducers of the inducible promoters which controltranscription of the agent. For example, in situ expression bygenetically modified cells of a therapeutic agent encoded by a geneunder the control of the metallothionein promoter, is enhanced bycontacting the genetically modified cells with a solution containing theappropriate (i.e., inducing) metal ions in situ.

Accordingly, the amount of therapeutic agent that is delivered in situis regulated by controlling such factors as: (1) the nature of thepromoter used to direct transcription of the inserted gene, (i.e.,whether the promoter is constitutive or inducible, strong or weak); (2)the number of copies of the exogenous gene that are inserted into thecell; (3) the number of transduced/transfected cells that areadministered (e.g., implanted) to the patient; (4) the size of theimplant (e.g., graft or encapsulated expression system); (5) the numberof implants; (6) the length of time the transduced/transfected cells orimplants are left in place; and (7) the production rate of thetherapeutic agent by the genetically modified cell. Selection andoptimization of these factors for delivery of a therapeuticallyeffective dose of a particular therapeutic agent is deemed to be withinthe scope of one of ordinary skill in the art without undueexperimentation, taking into account the above-disclosed factors and theclinical profile of the patient.

In addition to at least one promoter and at least one heterologousnucleic acid encoding the therapeutic agent, the expression vector mayinclude a selection gene, for example, a neomycin resistance gene, forfacilitating selection of cells that have been transfected or transducedwith the expression vector. Alternatively, the cells are transfectedwith two or more expression vectors, at least one vector containing thegene(s) encoding the therapeutic agent(s), the other vector containing aselection gene. The selection of a suitable promoter, enhancer,selection gene and/or signal sequence (described below) is deemed to bewithin the scope of one of ordinary skill in the art without undueexperimentation.

The therapeutic agent can be targeted for delivery to an extracellular,intracellular or membrane location. If it is desirable for the geneproduct to be secreted from the cells, the expression vector is designedto include an appropriate secretion “signal” sequence for secreting thetherapeutic gene product from the cell to the extracellular milieu. Ifit is desirable for the gene product to be retained within the cell,this secretion signal sequence is omitted. In a similar manner, theexpression vector can be constructed to include “retention” signalsequences for anchoring the therapeutic agent within the cell plasmamembrane. For example, all membrane proteins have hydrophobictransmembrane regions, which stop translocation of the protein in themembrane and do not allow the protein to be secreted. The constructionof an expression vector including signal sequences for targeting a geneproduct to a particular location is deemed to be within the scope of oneof ordinary skill in the art without the need for undue experimentation.

The following discussion is directed to various utilities of the instantdisclosure. For example, the instant disclosure has utility as anexpression system suitable for detoxifying intra- and/or extracellulartoxins in situ. By attaching or omitting the appropriate signal sequenceto a gene encoding a therapeutic agent capable of detoxifying a toxin,the therapeutic agent can be targeted for delivery to the extracellularmilieu, to the cell plasma membrane or to an intracellular location. Inone embodiment, the exogenous genetic material containing a geneencoding an intracellular detoxifying therapeutic agent, furtherincludes sequences encoding surface receptors for facilitating transportof extracellular toxins into the cell where they can be detoxifiedintracellularly by the therapeutic agent. Alternatively, the cells canbe genetically modified to express the detoxifying therapeutic agentanchored within the cell plasma membrane such that the active portionextends into the extracellular milieu. The active portion of themembrane-bound therapeutic agent detoxifies toxins, which are present inthe extracellular milieu.

In addition to the above-described therapeutic agents, some of which aretargeted for intracellular retention, the instant disclosure alsoembraces agents intended for delivery to the extracellular milieu and/oragents intended to be anchored in the cell plasma membrane.

The selection and optimization of a particular expression vector forexpressing a specific gene product in an isolated cell is accomplishedby obtaining the gene, potentially with one or more appropriate controlregions (e.g., promoter, insertion sequence); preparing a vectorconstruct comprising the vector into which is inserted the gene;transfecting or transducing cultured cells in vitro with the vectorconstruct; and determining whether the gene product is present in thecultured cells.

In certain embodiments, a virus from the adeno-associated virus familyis used. In certain embodiments, an expression vector for gene therapybased on AAV2, AAV4 and/or AAV5 is used.

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable viral expression vectors are available for transferringexogenous genetic material into cells. The selection of an appropriateexpression vector to express a therapeutic agent for a particularcondition amenable to gene replacement therapy and the optimization ofthe conditions for insertion of the selected expression vector into thecell, are within the scope of one of ordinary skill in the art withoutthe need for undue experimentation.

In an alternative embodiment, the expression vector is in the form of aplasmid, which is transferred into the target cells by one of a varietyof methods: physical (e.g., microinjection, electroporation, scrapeloading, microparticle bombardment) or by cellular uptake as a chemicalcomplex (e.g., calcium or strontium co-precipitation, complexation withlipid, complexation with ligand). Several commercial products areavailable for cationic liposome complexation including Lipofectin™(Gibco-BRL, Gaithersburg, Md.) and Transfectam™ (ProMega, Madison,Wis.). However, the efficiency of transfection by these methods ishighly dependent on the nature of the target cell and accordingly, theconditions for optimal transfection of nucleic acids into cells usingthe above-mentioned procedures must be optimized. Such optimization iswithin the scope of one of ordinary skill in the art without the needfor undue experimentation.

The instant disclosure also provides various methods for making andusing the above-described genetically-modified cells. As used herein,the term “isolated” means a cell or a plurality of cells that have beenremoved from their naturally-occurring in vivo location. Methods forremoving cells from a patient, as well as methods for maintaining theisolated cells in culture are known to those of ordinary skill in theart.

The instant disclosure also provides methods for genetically modifyingcells of a mammalian recipient in vivo. According to one embodiment, themethod comprises introducing an expression vector for expressing aheterologous gene product into cells of the mammalian recipient in situby, for example, injecting the vector into the recipient.

In one embodiment, the preparation of genetically modified cellscontains an amount of cells sufficient to deliver a therapeuticallyeffective dose of the therapeutic agent to the recipient in situ. Thedetermination of a therapeutically effective dose of a specifictherapeutic agent for a known condition is within the scope of one ofordinary skill in the art without the need for undue experimentation.Thus, in determining the effective dose, one of ordinary skill wouldconsider the condition of the patient, the severity of the condition, aswell as the results of clinical studies of the specific therapeuticagent being administered.

If the genetically modified cells are not already present in apharmaceutically acceptable carrier they are placed in such a carrierprior to administration to the recipient. Such pharmaceuticallyacceptable carriers include, for example, isotonic saline and otherbuffers as appropriate to the patient and therapy.

More than one recombinant gene can be introduced into each geneticallymodified cell on the same or different vectors, thereby allowing theexpression of multiple therapeutic agents by a single cell.

EXAMPLE 1 Treating Central Nervous System Disorders by Means of VascularEndothelia

Lysosomal storage disorders (LSDs) constitute a large class of inheritedmetabolic disorders. Most LSDs are caused by lysosomal enzymedeficiencies which lead to organ damage and often central nervous system(CNS) degeneration. Early work in rodent models of the lysosomal storagediseases (LSD) has shown promise in addressing the systemicmanifestations of these disorders, either by enzyme replacement or bonemarrow transplant to adult recipients. While enzyme replacement isefficacious for peripheral disease, treating the CNS remains a challengeas enzymes delivered intravenously do not cross the blood-brain barrier(BBB). Gene therapy studies in LSD animal models have thus far requireddirect intracranial injection of viral vectors. The cerebral vasculatureis an attractive target for gene therapy due to its extensive networkthroughout the brain that may potentially be co-opted to delivertherapeutic enzymes, but a vector that targets the vasculature is notavailable. Here the inventors used in vivo phage display to identifypeptides that bind to the vascular endothelia of a murine model ofmucopolysaccharidosis type VII (MPS VII), a prototypical LSD caused byβ-glucuronidase deficiency. In the β-glucuronidase deficient mouse,inhibition of cognitive decline required that treatment be initiated inthe neonatal period systemically prior to blood-brain barrier closure,or directly to brain. Insertion of the newly identified peptides intothe adeno-associated virus capsid resulted in virus that expressedtherapeutic enzyme from vascular endothelial cells. Importantly,intravenous injection of the modified virus rescued CNS deficits in theMPS VII mouse. These results demonstrate for the first time therapeuticefficacy based on retargeting viral tropism to a critical site ofdisease.

Prior studies have shown that soluble lysosomal enzymes are in partsecreted out of the cell, and can undergo mannose-6-phosphate receptormediated endocytosis and sorting to the lysosome by neighboring cells ina process termed cross correction. The inventors hypothesized thatexpression of lysosomal enzymes from brain vascular endothelia wouldlead to global cross correction of the brain by virtue of the densedistribution of CNS vasculature throughout the brain parenchyma. Thesurface area of the brain microvasculature is about 100 cm²/g of tissue.Transduction of vascular endothelial cells allows apical and basolateralsecretion of therapeutic agents such as β-glucuronidase. Thus, theinventors hypothesized that basolateral secretion can expose underlyingneurons and glia to recombinant enzyme sufficient for therapy (FIGS. 1Aand 1B). Currently, no AAVs target brain endothelium specifically orefficiently. Most AAVs are taken up by liver following peripheraldelivery.

The present inventors designed AAVs that are modified to target brainendothelia after systemic delivery. To generate an adeno-associatedvirus (AAV) that targets the cerebral vasculature, the inventors firstused in vivo phage display panning to identify peptide motifs that bindpreferentially to brain vasculature. A phage-display library wasinjected intravenously into wildtype and MPS VII mice, and the brainmicrovasculature was subsequently isolated along with the bound phage.The isolated phage was then amplified and reinjected, and after fiverounds of such in vivo panning, DNA sequencing of the recovered phagerevealed an enrichment of distinct peptide motifs from the initial phagelibrary. Interestingly, the motifs enriched in wildtype mice were alldistinct from those in MPSVII mice, suggesting a vascular remodelingprocess in the diseased mice. This method can be utilized to identifymotif(s) that are specific to other disease states.

In wildtype mice, the peptide motifs, PxxPS (SEQ ID NO: 1), SPxxP (SEQID NO: 2). TLH (SEQ ID NO: 3) and QSxY (SEQ ID NO: 4) were identified in19 of the clones (FIG. 2A). Of these, two peptides were especiallynotable, namely DSPAHPS (SEQ ID NO: 8) and GWTLHNK (SEQ ID NO: 16).DSPAHPS (SEQ ID NO: 15) contained both PxxPS (SEQ ID NO: 1) and SPxxP(SEQ ID NO: 2) motifs, and GWTLHNK (SEQ ID NO: 16) was represented 3times in the sampled phage population. In MPS VII mice, three peptidesmotifs—LxSS (SEQ ID NO: 21), PFxG (SEQ ID NO: 22) and SIxA (SEQ ID NO:23)—were identified (FIG. 2B). To confirm the affinity of these phagefor the brain vasculature, each phage was individually re-injectedintravenously into wildtype or MPS VII mice, and the amount of phagerecovered from brain vasculature was compared to that of either acontrol phage without a peptide insert, or the original unselected phagelibrary. In wildtype mice, the recovery of phage containing DSPAHPS (SEQID NO: 8) and GWTLHNK (SEQ ID NO: 16) was higher than the others (FIG.2C), consistent with the initial panning results. In MPS VII mice, thepeptides WPFYGTP (SEQ ID NO: 29) and LPSSLQK (SEQ ID NO: 25) were morehighly-recovered than the other selected phage (FIG. 2D). Consistentwith the panning results, each of the selected phage accumulated inbrain beyond the background levels observed for controls.

Peptide-modified AAVs were generated by inserting the peptidesidentified from phage display panning into the AAV2 capsid. Peptideswere inserted at position 587 of the VP3 capsid protein to yield clonesAAV-Linker(AAAAA (SEQ ID NO: 44)), AAV-TLH(GWTLHNK (SEQ ID NO: 16)),AAV-PPS(DSPAHPS (SEQ ID NO: 8)), AAV-PFG(WPFYGTP (SEQ ID NO: 29)) andAAV-LSS(LPSSLQK (SEQ ID NO: 25)). AAV-WT (no insert) served as a controlvirus. The 587 site is located in a domain of the VP3 capsid proteininvolved in the binding of AAV2 with its major receptor, heparin sulfateproteoglycan (HSPG), and insertion of peptides in this site can alterthe tropism of AAV without compromising virus viability. The modifiedcapsid proteins packaged AAV vector genomes with genomic titerscomparable to those of wildtype virus.

To assess the tissue tropism of the peptide-modified AAV, theinvestigators quantified viral genomes by RT-PCR in liver and brainfollowing tail vein injections of virus. AAV-PPS and AAV-TLH wereinjected into wildtype mice, and AAV-PFG and AAV-LSS were injected intoMPS VII mice. A design for peptide modified AAVs (PM-AAVs) is depictedin Scheme 1 below.

SCHEME 1 1. Sequence of AAV2 wild-type capsid            587 5885′-AGA GGC AAC AGA CAA GCA-3′ (SEQ ID NO: 36)    R   G   N   R   Q   A(SEQ ID NO: 37) 2. Modified sequence of capsid backbone               Not I           AscI5′-AGA GGC AAC GCG GCC GCC TAG GCG CGC CAA GCA-3′ (SEQ ID NO: 38)   R   G   N    A   A   A  stop A   R  Q   A (SEQ ID NO: 39)3. Insertion of peptide X into the NotI and AscI site   R   G   N    A   A   A  X    A   A  R   Q   N (SEQ ID NO: 40)

The sequence of AAV2 wild type capsid is depicted below in SEQ ID NO:35.An example of amino acids targeted to brain vasculature of MPS VII miceare italicized, and the underlined amino acids are spacers.

(SEQ ID NO: 35) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAA WPFYGTP AA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

AAV-WT was injected as a control in both mouse strains. At four weekspost-injection, AAV-WT transduced the liver predominantly, with no virusdetected in the brain of either wildtype or MPS VII mice after systemicadministration (FIGS. 3A and 3B). In contrast, intravenousadministration of peptide-modified AAV resulted in significant virusaccumulation in the brain and lower levels in the liver (FIGS. 3A and3B). The tropism of peptide-modified AAV was further confirmed byreporter gene expression. Peptide-modified AAV with the cytomegalovirus(CMV) promoter driving expression of either eGFP (AAV-PPS) orβ-glucuronidase (AAV-PFG) was injected via tail vein in wildtype and MPSVII mice, respectively. Consistent with the RT-PCR results, at 4 weekspost-injection, eGFP and β-glucuronidase expression was detected in thebrain of wildtype and MPS VII mice, respectively, and not observed inmice infused with AAV-WT. Furthermore, β-glucuronidase co-localized withCD31, a marker of vascular endothelium, confirming that the virus istargeting brain endothelium. The shift in tropism of these modified AAVis accompanied by a loss in affinity to heparin sulfate proteoglycan. Ina heparin-agarose binding assay, AAV-TLH, AAV-PPS, AAV-PFG and AAV-LSSall lost the ability to bind heparin sulfate. These results demonstratethat the peptides currently identified via phage display panningsuccessfully retargeted the tropism of AAV to the brain vascularendothelium.

Thus, in certain embodiments, an amino acid sequence that targets thevector to brain vascular endothelium is inserted, as discussed above. Incertain embodiments, that amino acid sequence may consist of, orcomprise, PXXPS (SEQ ID NO:1), SPXXP (SEQ ID NO:2), TLH (SEQ ID NO:3),QSXY (SEQ ID NO:4), LXSS (SEQ ID NO:21), PFXG (SEQ ID NO:22), or SIXA(SEQ ID NO:23), as expressed in an amino to carboxy orientation or in acarboxy to amino orientation. In certain embodiments, that sequence mayconsist of, or comprise, PYFPSLS (SEQ ID NO:5), YAPLTPS (SEQ ID NO:6),PLSPSAY (SEQ ID NO:7), DSPAHPS (SEQ ID NO:8), GTPTHPS (SEQ ID NO:9),PDAPSNH (SEQ ID NO:10), TEPHWPS (SEQ ID NO:11), SPPLPPK (SEQ ID NO:12),SPKPPPG (SEQ ID NO:13), NWSPWDP (SEQ ID NO:14), DSPAHPS (SEQ ID NO:15),GWTLHNK (SEQ ID NO:16), KIPPTLH (SEQ ID NO:17), ISQTLHG (SEQ ID NO:18),QSFYILT (SEQ ID NO:19), TTQSEYG (SEQ ID NO:20), MLVSSPA (SEQ ID NO:24),LPSSLQK (SEQ ID NO:25), PPLLKSS (SEQ ID NO:26), PXKLDSS (SEQ ID NO:27),AWTLASS (SEQ ID NO:28), WPFYGTP (SEQ ID NO:29), GTFPFLG (SEQ ID NO:30),GQVPFMG (SEQ ID NO:31), ANFSILA (SEQ ID NO:32), GSIWAPA (SEQ ID NO:33),or SIAASFS (SEQ ID NO:34), as expressed in an amino to carboxyorientation or in a carboxy to amino orientation.

Delivery of therapeutic enzymes to the central nervous system continuesto be a major challenge in treating neuronopathic lysosomal storagedisorders as the blood brain barrier effectively prevents entry ofenzymes from the systemic circulation. Here the investigators tested thetherapeutic efficacy of targeting brain vascular endothelia with themodified AAV in the murine model of MPS VII. Thisβ-glucuronidase-deficient mouse exhibits hallmarks of MPS VII diseaseincluding lysosomal storage, and neurological dysfunction, and is aproven model for investigating novel therapies for lysosomal storagedisorders (Vogler, C. et al., Pediatr Res 49, 342-8 (2001)). AAV-PFG orAAV-WT expressing β-glucuronidase was injected via tail vein in the MPSVII mice at 6 weeks of age, a time when lysosomal storage deposits firstappear in the mouse. At 6 weeks post-injection, lysosomal storage andcellular distension in the brain was investigated via bright-fieldmicroscopy. MPS VII mice treated with AAV-PFG exhibited reduced levelsof lysosomal storage relative to mice receiving AAV-WT, which retainedlysosomal storage in multiple regions of the CNS, including cerebralcortex, hippocampus, striatum, and cerebellum (FIGS. 4A-4H). Because thepresent peptide-modified viruses specifically target endothelia, thecorrection of neuronal pathology suggests that β-glucuronidase issecreted basolaterally by endothelial cells and subsequentlycross-correcting adjacent neurons. The correction of neuropathology inmultiple structures throughout the entire rostral-caudal extent of thebrain indicates a broad dissemination of therapeutic enzyme.

MPS VII mice develop progressive impairment of neuronal function asmeasured by Morris water maze, repeated acquisition and performancechamber (RAPC), and context fear-conditioning assays. To test forfunctional recovery after intravenous delivery of PM-AAV, the presentinvestigators used the context fear-conditioning assay, which tests theintegrity of several brain regions including the hippocampus and theamygdala. Mice were first conditioned by foot-shocks in the testingchamber (context 1). One day later, fear-induced freezing behavior wasmeasured when the mice were placed either in context 1 or a modifiedchamber with novel olfactory, tactile, and visual cues (context 2).Control mice were able to distinguish context 1 from context 2, asevidenced by the decrease in freezing behavior when placed in context 2.MPS VII mice treated with AAV-WT-β-glucuronidase, in contrast, exhibitedless change in freezing behavior, suggesting the persistence of memorydeficits. MPS VII mice treated with AAV-PFG-β-glucuronidase, however,exhibited behavior similar to that of heterozygous mice (FIG. 4I).Intravenous injection of PM-AAV, and subsequent expression ofβ-glucuronidase from brain endothelia, rescued these CNS deficits of theMPS VII mouse.

The deficient enzyme in MPS VII, β-glucuronidase, catalyzes thedegradation of glycosaminoglycans (GAG's) including heparin sulfate andchondroitin sulfate. In the disease state, catabolism of these moleculesis blocked and results in lysosomal accumulation. The investigatorshypothesized that peptide-modified AAV might interact withGAG-containing glycoproteins that accumulate on endothelial surfaces. Toaddress this hypothesis, the ability of AAV-PFG to bind to purifiedbrain vasculature from MPS VII mice in the presence and absence of theenzyme chondroitinase ABC was measured. Enzymatic treatment of thevasculature from MPS VII mice abolished the binding ability to thePFG-AAV (FIG. 4J). It was further demonstrated that an excess ofchondroitin sulfate in the binding reaction was able to compete away thebinding of PFG-AAV to the vasculature (FIG. 4K). These results suggestthat the binding of the present modified virus (AAV-PFG) to the brainvascular endothelia requires chondroitin sulfate.

As proof of principle that the success of the panning experiments isapplicable to disease models beyond the MPS VII mouse, the presentinvestigators carried out the same experiment in a mouse model of lateinfantile neuronal ceroid lipofuscinosis. This mouse lacks expression ofthe lysosomal enzyme tripeptidyl peptidase I (TPP1), and recapitulatesmany pathological features of the human disease. After five rounds of invivo panning, a single dominant peptide emerged-GMNAFRA (SEQ ID NO:41)(FIG. 5A). As before, a peptide-modified AAV expressing TPP1 wasproduced and injected intravenously in TPP1-deficient mice. Three weekspost-injection, mice that received PM-AAV exhibited TPP1 expression insmall vessels of the cerebral cortex, midbrain, and cerebellum, whereasmice that received injections of wildtype AAV did not show any TPP1staining (FIGS. 5B-5D). The peptide identified in this experiment wasdistinct from those in wildtype or MPS VII mice, again suggesting adisease-specific vascular remodeling process. An in vitro assay for TPP1activity in several tissues following tail vein injection of peptidemodified virus was also performed (FIG. 5E). Activity levels expressedrelative to heterozygous control.

The ability to systematically alter viral tropism has emerged as apowerful technique for gene therapy. Here the present investigatorsretargeted AAV to the brain vasculature as a means to disseminatetherapeutic enzyme, and demonstrated for the first time a correction ofCNS disease in the MPS VII mouse following peripheral delivery of genetherapy vectors.

Thus, peripheral delivery of peptide-modified AAVs targeted to the braintreated pathology and improved behavioral deficits when delivered toadult mice with established disease. Modified vectors, e.g.,peptide-modified AAVs, can be used in therapies for the CNS aspects ofthe LSDs.

The inventors also studied the in vivo biodistribution of peptidemodified AAV. Peptide-modified AAV (1.0×10¹¹ GPs) were injectedintravenously into mice, with AAV-PPS and AAV-TLH to wild type mice,AAV-PFG and AAV-LSS to MPS VII mice. After 4 weeks, mice were killed,brain and liver were harvested and genomic DNA was extracted. The virusbiodistribution was assessed by real-time PCR. Also, AAV-PFG-βGluc orAAV-WT-βGluc (10×10¹² gp/ml) was injected into the mice through tailvein. Six weeks later, the serum was isolated and analysis ofβ-glucuronidase activity by fluorescent substrate assay was performed.The results showed that enzyme delivered to brain was also reaching theperiphery.

Materials and Methods:

Experimental animals: MPS VII (B6.C-H-2^(bm1)/byBir-gus^(mps)/+) miceand heterozygous controls were obtained from the Jackson Laboratory (BarHarbor, Me.), and subsequently bred and maintained at the University ofIowa animal facility. TPP1-deficient (CLN2−/−) mice have been describedpreviously. All animal maintenance conditions and experimental protocolswere approved by the University Of Iowa Animal Care and Use Committee.

In vivo biopanning: MPS VII and wild type mice (6-8 weeks of age) wereeach injected through the tail vein with 2×10¹⁰ pfu of phage from thePh.D.™-7 phage display library (New England Biolabs®, Ipswich, Mass.) in200 μl DMEM (Invitrogen™, Carlsbad, Calif.) through the tail vein. Afterincubation for five minutes, the mice were anesthetized and perfusedtranscardially with DMEM. The brain was then extracted, and the bindingphage was recovered and amplified. The amplified phage was thenpurified, titered, and re-injected in each of five consecutive rounds ofpanning. The selected phage and phage control (no inserted peptide) wereamplified individually. The original Ph.D.™-7 phage display library wasused as unselected control. The input phage was kept at 2×10¹⁰ pfu/micein each round. After the fifth round of panning, DNA from 20 randomlyselected phage clones was sequenced with the primer-96gIII (New EnglandBiolabs®, Ipswich, Mass.).

Construction of peptide modified AAV2 capsids: The plasmid for cloningof modified capsids was developed from pXX2, containing the wild-typeAAV2 Rep and Cap. A plasmid with a DNA fragment encoding amino acidsAAAstopA and the restriction sites NotI and AscI inserted between AAV2Cap amino acid position 587 and 588 was constructed as the backboneplasmid. dsDNA inserts encoding selected peptides were cloned into NotIand AscI site as peptide modified pXX2.

AAV2 production and titer: Plates of 293T cells were cotransfected withthree plasmids: pXX2 or peptide modified pXX2, which supplied the Repand Cap proteins of AAV2; pHelper, which contained the adenovirus helperfunctions; and a vector plasmid, which contained the AAV2 ITRs and thetransgene of interest. Twenty 150 mm-diameter plates were cotransfected90 μg DNA of plasmids pXX2, pHelper, and vector at a molar ratio of1:1:1. After incubating for 60 hours, the virus was purified withiodixanol gradients and further purification through a mustang Qmembrane. Titers of recombinant AAV were determined by real-time PCR.

In vitro heparin binding assay: Viral particles (1.0×10¹⁰ genomeparticles) were bound to 50 μl of heparin agarose in 1 mlphosphate-buffered saline containing 1 mM MgCl₂ and 2.5 mM KCl (PBS-MK)for 2 hr at 4° C. with gentle mixing. This was then washed three timeswith 1 ml PBS-MK and then eluted with 30 μl PBS-MK containing 2M NaClwith vigorous vortexing. Eluted samples were analyzed by western blotwith anti-AAV antibody.

In vivo biodistribution of virus: 6 to 8 weeks-old MPS VII andage-matched wildtype control mice were injected intravenously with1.0×10¹¹ genome particles of wild type AAV2 or peptide modified AAV2(PM-AAV) via the tail vein (n=3 mice per experimental group). At 4 weekspost-injection, mice were sacrificed and tissues were harvested and snapfrozen. Genomic DNA from representative organs was extracted using aQiagen® DNA extraction kit. AAV copies in a particular organ weredetermined by real-time PCR.

In situ enzyme activity assay: Mice injected with AAV were anesthetizedand transcardially perfused with ice-cold 2% paraformaldehyde 4 weekspost-injection. Brains were harvested, embedded in OCT compound, andsectioned (16 μm) on a cryostat. Sections were washed in 0.05M NaOAC, pH4.5 at 4° C.×10 min, incubated in 0.25 mM naphthol-SD-BI-β-D-glucuronidein 0.05M NaOAC at 37° C.×40 min, and then stained at 37°×2-4 hrs with0.25 mM naphthol-SD-BI-β-D-glucuronidein 0.05M NaOAc buffer, pH5.2 with1/500 2% hexazotized pararosaniline. Sections were counterstained with0.5% methyl green solution.

MPS VII Histology: Mice were transcardially perfused with 2%paraformaldehyde and 2% glutaraldehyde in PBS, then post-fixed in thesame fixative at 4° C. overnight. Tissues were blocked, fixed in 2.5%glutaraldehye for 1 hour at room temperature and then post fixed in 1%OsO4 for 2 hours at room temperature. Samples were then dehydrated andembedded in Epon™ compound. 1 μm thick sections were stained withtoluidine blue solution and analyzed for cell morphology using anOlympus BX-51 Digital Light Microscope.

TPP1 Immunostaining: Mice were deeply anesthetized with intraperitonealketamine (100-125 mg/kg) and xylazine (10-12.5 mg/kg), thentranscardially perfused with normal saline (20 mL) followed by 4%paraformaldehyde in normal saline (20 mL). Brains were then extractedand post-fixed in 4% paraformaldehyde for 24 hours at 4° C. 40 μm thicksections were cut on a freezing microtome and collected free floating incryoprotectant solution (30% ethylene glycol, 15% sucrose, 0.05% sodiumazide, in TBS) for storage at −20° C. Free floating sections wereimmunostained with anti-TPP1 primary antibody (Abcam) diluted in TBSwith 2% BSA, 0.1% NaN₃, and 0.05% Tween 20. After incubation withprimary antibody overnight at 4° C., tissue sections were rinsed withTBS and incubated with biotinylated goat anti-mouse secondary antibody(Jackson). Stains were developed with DAB peroxidase substrate kit(Vector Laboratories).

Context fear conditioning: The experiments were performed as previouslydescribed (Liu, G. et al., J Neurosci 25, 9321-7 (2005)). Six weeksafter intravenous injection with 1×10¹² genome particles of virus, mice(n=6 per group) were subjected to testing in a fear-conditioning chamber(Med Association, San Diego, Calif.). Briefly, after 3 min ofacclimation to the testing chamber (context 1), each mouse receivedseven successive electrical foot shocks (2 min apart, 0.75 mA, 50 Hz, 1sec duration). Fear response was determined by measuring the amount offreezing behavior, which was defined as the lack of any movement otherthan respiratory activity. Freezing in the first 3 min after placementinto the chamber was recorded during the training and again 24 h laterin two contexts. Context 1 was the one used for training. Context 2 wasa modified chamber with new olfactory, tactile, and visual cues. Animalsstayed in their home cages during the 2 h interval between tests in thetwo contexts.

Analysis of PM-AAV binding site: Mouse brain vasculature was isolated bycentrifuging crude brain homogenate in 15% Dextran, and further purifiedby running through 105 and 70 μm meshes. The vasculature separated bythe 70 μm mesh were used. 50 mg of brain vasculature was incubated withPBS alone, PNGase (100 U/reaction), or chondroitinase ABC (2 U/reaction)at 37° C.×1 hr. The reaction was stopped by adding cold PBS and washed 3times. The treated vasculatures were then incubated with virus(1.0×10¹¹) in 500 μl PBS with 0.1% BSA at 4° C.×1 hr. After washing, theDNA was isolated and viral genomic particles were analyzed by real timePCR. For competitive binding of AAV-PFG to brain vasculature of MPS VIImice, 50 mg of brain vasculatures were incubated with AAV-PFG (1.0×10¹¹)in the presence or absence of 2 mg/ml chondroitin sulfate at 4° C.×1 hr.

Statistical analysis: All data are expressed as means±standarddeviation. An unpaired student t-test was applied to test forstatistical differences. Data were considered significant when p<0.05.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A modified adeno-associated virus (AAV) capsid protein comprising atargeting peptide, wherein the targeting peptide is from 3 to 10 aminoacids in length and wherein the targeting peptide targets an AAV tobrain vascular endothelium, wherein the targeting peptide is GMNAFRA(SEQ ID NO:41), as expressed in an amino to carboxy orientation or in acarboxy to amino orientation.
 2. The capsid protein of claim 1, whereinthe targeting peptide targets a diseased brain vascular endothelium. 3.The capsid protein of claim 2, wherein the target peptide targets brainvascular endothelium in a subject that has a lysosomal storage disease.4. The capsid protein of claim 2, wherein the targeting peptide targetsTPP1-deficient-brain vascular endothelium.
 5. The capsid protein ofclaim 1, wherein the AAV is AAV2.
 6. An AAV virus containing the capsidprotein of claim
 1. 7. A method to deliver an agent to the centralnervous system of a subject, comprising administering the virus of claim6 to the subject.
 8. The method of claim 7, wherein the AAV is AAV2. 9.The AAV virus of claim 6, wherein the AAV is AAV2.